From Ganglion Cell to Photoreceptor Layer: Timeline of Deterioration in a Rat Ischemia/Reperfusion Model

Neuronal damage and impaired vision in different retinal disorders are induced, among other factors, by ischemia/reperfusion (I/R). Since the mechanisms and the progression of ischemic injury are still not completely clarified, a timeline of this retinal degeneration is needed. In this study, we investigated protein and mRNA alterations at 2, 6, 12, and 24 h as well as 3 and 7 days after ischemia to determine the course of an ischemic insult through the whole retina. Moreover, functional analyses were performed at later stages. We detected a significant functional loss of cells in the inner nuclear layer and photoreceptors at 3 and 7 days. Additionally, the thickness of the whole retina was decreased at these points in time, indicating a severe degradation of all retinal layers. Immunohistological and qRT-PCR analyses of retinal ganglion cells (RGCs), glial cells, AII amacrine, cone and rod bipolar as well as cone and rod photoreceptor cells confirmed this first assumption. Our results show that all investigated cell types were damaged by ischemia induction. Especially RGCs, cone bipolar cells, and photoreceptor cones are very sensitive to I/R. These cells were lost shortly after ischemia induction with a progressive course up to 7 days. In addition, Müller cell gliosis was observed over the entire period of time. These results provide evidence, that I/R induces damage of the whole retina at early stages and increases over time. In conclusion, our study could demonstrate the intense impact of an ischemic injury. The ischemic defect spreads across the whole retina right up to the outer layers in the long-term and thus seems to impair the visual perception already during the stimulus processing. In addition, our findings indicate that the cone pathway seems to be particularly affected by this damage

Comparable effects on tear film parameters after femtosecond laser-assisted and conventional cataract surgery

Purpose!#!Dry eye symptoms after conventional cataract surgery are a very common problem. Until now, only few data are available on objective tear film parameters in regard to femtosecond laser-assisted cataract surgery (LCS). Aim of this study was therefore to analyze and compare tear film parameter changes between LCS and conventional cataract surgery.!##!Methods!#!A consecutive group of 34 patients, scheduled for cataract surgery, were randomly selected for either LCS or conventional cataract surgery (17 patients/group). Tear film assessments including tear film osmolarity, Schirmer test, MMP-9 analysis via quantitative ELISA, corneal sensitivity, corneal fluorescein staining, and conjunctival fluorescein staining were sequentially evaluated pre- as well as 1 and 3 months postoperatively.!##!Results!#!Both groups showed no significant difference in baseline characteristics. All surgeries were performed without any complications. After 1 and 3 months, there was no statistically significant difference in regard to tear film osmolarity (1 month: p = 0.81, 3 months: p = 1.0), Schirmer test (1 month: p = 0.35, 3 month: p = 0.08), and MMP-9 concentration (1 month: p = 0.36, 3 month: p = 0.28) between the two groups.!##!Conclusions!#!Neither LCS nor conventional cataract surgery affected objective tear film parameters significantly during our 3-month postoperative observation period. Hence, both surgical techniques can be equally used to treat patients without prior dry eye symptoms

Fewer Functional Deficits and Reduced Cell Death after Ranibizumab Treatment in a Retinal Ischemia Model

Retinal ischemia is an important factor in several eye disorders. To investigate the impact of VEGF inhibitors, as a therapeutic option, we studied these in a retinal ischemia animal model. Therefore, animals received bevacizumab or ranibizumab intravitreally one day after ischemia induction. Via electroretinography, a significant decrease in a- and b-wave amplitudes was detected fourteen days after ischemia, but they were reduced to a lesser extent in the ranibizumab group. Ischemic and bevacizumab retinae displayed fewer retinal ganglion cells (RGCs), while no significant cell loss was noted in the ranibizumab group. Apoptosis was reduced after therapy. More autophagocytotic cells were observed in ischemic and bevacizumab eyes, but not in ranibizumab eyes. Additionally, more microglia, as well as active ones, were revealed in all ischemic groups, but the increase was less prominent under ranibizumab treatment. Fewer cone bipolar cells were detected in ischemic eyes, in contrast to bevacizumab and ranibizumab-treated ones. Our results demonstrate a reduced apoptosis and autophagocytosis rate after ranibizumab treatment. Furthermore, a certain protection was seen regarding functionality, RGC, and bipolar cell availability, as well as microglia activation by ranibizumab treatment after ischemic damage. Thus, ranibizumab could be an option for treatment of retinal ischemic injury

Specific inner retinal layer cell damage in an autoimmune glaucoma model is induced by GDNF with or without HSP27

PURPOSE: Previously, immunization of rats with ocular antigens induced retinal ganglion cell (RGC) degeneration. We investigated the effect of immunization with glial cell line-derived neurotrophic factor (GDNF) or GDNF in combination with heat shock protein 27 (GDNF+HSP) on RGCs and other retinal cells. METHODS: Rats were immunized with GDNF or GDNF+HSP. After 4 weeks, retinas were stained with Brn-3a and NeuN to quantify RGCs. GFAP and vimentin staining were used to investigate macroglia. Microglia were marked with Iba1 and ED1. Amacrine cells were labeled with parvalbumin and ChAT. Photoreceptors were evaluated with rhodopsin and opsin staining and bipolar cells with PKCα and recoverin. For these cell types, Western blotting was also performed. RESULTS: Retinas of immunized animals showed a significant loss of Brn-3a+ and NeuN+ RGCs. No significant changes could be observed in regard to macroglia. An increase in Iba1+ microglia was detected in both groups, but little change in regard to activated microglia. A loss of cholinergic amacrine cells was seen in the GDNF+HSP group by immunohistochemistry and in both groups via Western blot analysis. AII amacrine cells, bipolar cells, and photoreceptors were not affected. CONCLUSIONS: Immunizations led to loss of RGCs and cholinergic amacrine cells and a strong increase in microglial cells. Our data suggest that RGC loss is the consequence of immunization with GDNF. Astrocyte activity and its neuroprotective effects seem to be inhibited by GDNF immunization. We presume more complex interactions between GDNF and HSP27 because no additive effects could be observed

Follicular flushing in natural cycle IVF does not affect the luteal phase - a prospective controlled study.

In contrast to multifollicular IVF, follicular flushing seems to increase the efficacy of monofollicular IVF treatments such as natural cycle IVF (NC-IVF). However, because follicular flushing causes loss of granulosa cells, it might negatively affect luteal phase length and endocrine function of the luteal body. A prospective cohort Phase II study was performed in 24 women undergoing NC-IVF. Women underwent a reference cycle with human chorionic gonadotrophin-induced ovulation without follicle aspiration and analysis of the length of the luteal phase and luteal concentrations of progesterone and oestradiol. In addition, they underwent a NC-IVF cycle which was performed identically but follicles were aspirated and flushed three times. The luteal phase was shorter in 29.2%, equal in 16.7% and longer in 50.0% of cases following flushing of the follicles. Overall, neither difference in luteal phase length was significant [median duration (interquartile range) in reference cycle: 13 (12; 14.5), IVF (flushing) cycle: 14 (12.5; 14.5), median difference (95% CI): 0.5 (-0.5 to 1.5)] nor median progesterone and oestradiol concentrations. In conclusion, follicular flushing in NC-IVF affects neither the length of the luteal phase nor the luteal phase concentrations of progesterone and oestradiol, questioning the need for luteal phase supplementation

Anti-inflammatory cytokine and angiogenic factors levels in vitreous samples of diabetic retinopathy patients.

Evaluation of cytokines in patients with diabetic retinopathy (DR) is important for the identification of future additive or alternative treatment options. Therefore, vitreous samples were obtained from patients with DR and patients with macular hole or macular pucker (control group) during 23-gauge-vitrectomy (n = 17/group). The levels of three pro-inflammatory (IL-1ß, IL-6, IFN-γ) and pleiotropic cytokines (IL-2, IL-4, IL-13) as well as VEGF, VEGF-A, and PGF were measured using an enzyme linked immunosorbent assay (ELISA). IL-1ß (p = 0.02) and IFN-γ (p = 0.04), two of the three tested pro-inflammatory cytokines, were elevated in the DR patients, while IL-6 (p = 0.51) level was comparable in both groups. Moreover, in DR samples, a trend towards an IL-13 down-regulation (p = 0.36) was observable. The IL-2 (p = 0.62) and IL-4 (p = 0.78) levels were comparable in both groups. All analyzed angiogenetic factors were up-regulated in DR patients (VEGF: p<0.001; VEGF-A: p = 0.002; PGF: p<0.001). The up-regulation of angiogenetic factors underline their importance in DR development. However, the interaction of the other cytokines showed an interesting pattern. Pro-inflammatory cytokines were also up-regulated, which could be evidence for inflammation processes in the diabetic retina. Furthermore, it seems that a counter response of immunomodulatory cytokines is in an initial process, but not strong enough to regulate the processes. Therefore, to support these anti-inflammatory mechanisms might be additive treatment option in the future

Optic Nerve Degeneration after Retinal Ischemia/Reperfusion in a Rodent Model

Retinal ischemia is a common pathomechanism in many ocular disorders such as age-related macular degeneration (AMD), diabetic retinopathy, glaucoma or retinal vascular occlusion. Several studies demonstrated that ischemia/reperfusion (I/R) leads to morphological and functional changes of different retinal cell types. However, little is known about the ischemic effects on the optic nerve. The goal of this study was to evaluate these effects. Ischemia was induced by raising the intraocular pressure (IOP) in one eye of rats to 140 mmHg for 1 h followed by natural reperfusion. After 21 days, histological as well as quantitative real-time PCR (qRT-PCR) analyses of optic nerves were performed. Ischemic optic nerves showed an infiltration of cells and also degeneration with signs of demyelination. Furthermore, a migration and an activation of microglia could be observed histologically as well as on mRNA level. In regard to macroglia, a trend toward gliosis could be noted after ischemia induction by vimentin staining. Additionally, an up-regulation of glial fibrillary acidic protein (GFAP) mRNA was found in ischemic optic nerves. Counting of oligodendrocyte transcription factor 2 positive (Olig2+) cells revealed a decrease of oligodendrocytes in the ischemic group. Also, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) mRNA expression was down-regulated after induction of I/R. On immunohistological level, a decrease of MOG was detectable in ischemic optic nerves as well. In addition, SMI-32 stained neurofilaments of longitudinal optic nerve sections showed a strong structural damage of the ischemic optic nerves in comparison to controls. Consequently, retinal ischemia impacts optic nerve degeneration. These findings could help to better understand the course of destruction in the optic nerve after an ischemic insult. Especially for therapeutic studies, the optic nerve is important because of its susceptibility to be damaged as a result to retinal ischemic injury and also its connecting function between the eye and the brain. So, future drug screenings should target not only the retina, but also the functionality and structure of the optic nerve. In the future, these results could lead to the development of new therapeutic strategies for treatment of ischemic injury

Activation of Apoptosis in a βB1-CTGF Transgenic Mouse Model

To reveal the pathomechanisms of glaucoma, a common cause of blindness, suitable animal models are needed. As previously shown, retinal ganglion cell and optic nerve degeneration occur in βB1-CTGF mice. Here, we aimed to determine possible apoptotic mechanisms and degeneration of different retinal cells. Hence, retinae were processed for immunohistology (n = 5–9/group) and quantitative real-time PCR analysis (n = 5–7/group) in 5- and 10-week-old βB1-CTGF and wildtype controls. We noted significantly more cleaved caspase 3+ cells in βB1-CTGF retinae at 5 (p = 0.005) and 10 weeks (p = 0.02), and a significant upregulation of Casp3 and Bax/Bcl2 mRNA levels (p +) cells were detected in transgenic mice at 5 (p = 0.03) and 10 weeks (p = 0.02). Neurofilament H staining (p = 0.01) as well as Nefh (p = 0.02) and Tubb3 (p = 0.009) mRNA levels were significantly decreased at 10 weeks. GABAergic synapse intensity was lower at 5 weeks, while no alterations were noted at 10 weeks. The glutamatergic synapse intensity was decreased at 5 (p = 0.007) and 10 weeks (p = 0.01). No changes were observed for bipolar cells, photoreceptors, and macroglia. We conclude that apoptotic processes and synapse loss precede neuronal death in this model. This slow progression rate makes the βB1-CTGF mice a suitable model to study primary open-angle glaucoma

Protective effects on the retina after ranibizumab treatment in an ischemia model.

Retinal ischemia is common in eye disorders, like diabetic retinopathy or retinal vascular occlusion. The goal of this study was to evaluate the potential protective effects of an intravitreally injected vascular endothelial growth factor (VEGF) inhibitor (ranibizumab) on retinal cells in an ischemia animal model via immunohistochemistry (IF) and quantitative real-time PCR (PCR). A positive binding of ranibizumab to rat VEGF-A was confirmed via dot blot. One eye underwent ischemia and a subgroup received ranibizumab. A significant VEGF increase was detected in aqueous humor of ischemic eyes (p = 0.032), whereas VEGF levels were low in ranibizumab eyes (p = 0.99). Ischemic retinas showed a significantly lower retinal ganglion cell number (RGC; IF Brn-3a: p<0.001, IF RBPMS: p<0.001; PCR: p = 0.002). The ranibizumab group displayed fewer RGCs (IF Brn-3a: 0.3, IF RBPMS: p<0.001; PCR: p = 0.007), but more than the ischemia group (IF Brn-3a: p = 0.04, IF RBPMS: p = 0.03). Photoreceptor area was decreased after ischemia (IF: p = 0.049; PCR: p = 0.511), while the ranibizumab group (IF: p = 0.947; PCR: p = 0.122) was comparable to controls. In the ischemia (p<0.001) and ranibizumab group (p<0.001) a decrease of ChAT+ amacrine cells was found, which was less prominent in the ranibizumab group. VEGF-receptor 2 (VEGF-R2; IF: p<0.001; PCR: p = 0.021) and macroglia (GFAP; IF: p<0.001; PCR: p<0.001) activation was present in ischemic retinas. The activation was weaker in ranibizumab retinas (VEGF-R2: IF: p = 0.1; PCR: p = 0.03; GFAP: IF: p = 0.1; PCR: p = 0.015). An increase in the number of total (IF: p = 0.003; PCR: p = 0.023) and activated microglia (IF: p<0.001; PCR: p = 0.009) was detected after ischemia. These levels were higher in the ranibizumab group (Iba1: IF: p<0.001; PCR: p = 0.018; CD68: IF: p<0.001; PCR: p = 0.004). Our findings demonstrate that photoreceptors and RGCs are protected by ranibizumab treatment. Only amacrine cells cannot be rescued. They seem to be particularly sensitive to ischemic damage and need maybe an earlier intervention